The purpose of this project is to elucidate the gene structure and sequence of a non-muscle myosin. This project is part of the general effort in the Lab of Cell Biology to understand the organization and function of the cytoskeleton, using as a model system the soil amoeba Acanthamoeba. Acanthamoeba expresses simultaneously at least three distinct myosin enzymes, myosin IA, myosin IB and myosin II. Using molecular cloning techniques, we have isolated and purified a myosin II heavy chain gene and a myosin IB heavy chain gene. These genes were detected in a genomic library of Acanthamoeba DNA using a heterologous myosin gene (C. elegans) as a probe. the amoeba genomic clones were identified as myosin heavy chain genes by hybrid selection of myosin messenger RNA, in vitro translation, and specific immunoprecipitation. We are currently seeking to define the basic organization of these genes and to obtain sequence data from regions of the myosins which should be highly conserved. We intend to sequence these genes in their entirety. The significance of this work is that it will provide for the first time the sequence of a non-muscle myosin. While non-muscle and muscle myosins share many common features, non-muscle myosins do possess unique structural, enzymatic, and regulatory properties. The amoeba myosin sequence data will be of great value in furthering our understanding of the unique structural and functional aspects of the amoeba myosins. From the sequence data we will synthesize peptides, generate antibodies against these peptides, and use these antibodies as tools to analyze the function of the amoeba myosins in in vitro motility assays. We will then use these techniques to generate cytoskeletal proteins (myosins and/or other important cytoskeletal proteins) which have altered primary structure as a tool for dissecting the interrelationship between structure and function at the protein level.